The mechanism of DNA packaging for double-stranded DNA viruses will be studied in the Bacillus subtilis bacteriophage X29, the most efficient in vitro viral packaging system known. Using an integrated genetic, biochemical and structural approach, we will characterize protein conformational change and movement in the transiently assembled packaging motor during DNA encapsidation. The mechanism of packaging in X29 will serve as a model for animal virus packaging in the analogous herpesvirus and adenovirus systems, and aid in the search for new antiviral therapies. Due to similarities between the X29 ATPase and other ring translocases, insights gained from the study of X29 packaging will also provide insight into the basic principles of macromolecular motor function in higher organisms. To elucidate the mechanism of DNA packaging: an atomic structure of the X29 DNA packaging motor will be obtained by fitting of X-ray crystallographic structures of motor components into high-resolution cryoEM maps of the motor complex (Aim 1); high-resolution cryoEM reconstruction of packaging intermediates will identify mobile elements during DNA packaging (Aim 2); mutagenesis and biochemical analysis will dissect functional residues in the ATPase and connector that are crucial for DNA packaging (Aim 3); and, packaging motors containing chimeric pRNAs will be created to probe the global assembly, operation and communication of the X29 DNA packaging motor (Aim 4).